WO2004070024A1 - Mutants de tyrosyl t-arn synthase, et procede de construction de ceux-ci - Google Patents

Mutants de tyrosyl t-arn synthase, et procede de construction de ceux-ci Download PDF

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WO2004070024A1
WO2004070024A1 PCT/JP2004/001441 JP2004001441W WO2004070024A1 WO 2004070024 A1 WO2004070024 A1 WO 2004070024A1 JP 2004001441 W JP2004001441 W JP 2004001441W WO 2004070024 A1 WO2004070024 A1 WO 2004070024A1
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thigh
tyrrs
tyrosine
amino acid
trna
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PCT/JP2004/001441
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Japanese (ja)
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Shigeyuki Yokoyama
Kensaku Sakamoto
Osamu Nureki
Takatsugu Kobayashi
Masahiro Takahashi
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Riken
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the present invention relates to a tyrosyl-tRNA synthetase (hereinafter, referred to as TyrRS) and a Z-tyrosine tRNA (hereinafter, referred to as TyrRS) of Methanococcus jannasi; hereinafter, referred to as M. zo ⁇ ? /? A force 77.
  • TyrRS tyrosyl-tRNA synthetase
  • TyrRS Z-tyrosine tRNA
  • the modified aaRS (aaRS *) must first have the property of covalently binding unnatural amino acids to the modified tRNA (tRNA *).
  • tRNA * must not pair with 61 types of sense codons, but must have the property of pairing with other codons, such as amber codon [Wang et al., Chemical Co. unicat ions, 2002 1-11 (Reference 9)].
  • aaRS * does not amylate other than tRNA * and that tRNA * is not aminoacylated other than aaRS *.
  • a method of introducing pairs of other species into the host system has been adopted. The first attempt was to introduce the phenylalanyl-tRNA synthetase and tRNA Phe pair of budding yeast into Escherichia coli, and to introduce 7 "fluorophenylalanine specifically to ambercodon [Furter, Protein Science Vol. 7, 1998. Pp.
  • the archaebacterium j Fiber aschi i ⁇ ⁇ tRNA Tyi "pair is an Escherichia coli system
  • the E. coli Tyr RS and 3 c / / / 5 / ea 0 thermoph ilus tRNA Tyr are mammalian cell systems. Were used to extend their artificial genetic code [Ref 15 and Wang et al., Lournal of the American Chemical Society, Vol. 122, 2000, p. 50 10-50 11 (Ref. 19)].
  • TyrRS identifies tRNAs by a partial nucleotide sequence called an identity determinant possessed by the corresponding tRNA [Giege et al., Nucleic Acids Research, 26, 1998, p. 5017-5035 (Reference 20) )].
  • Archaeal and eukaryotic tRNAs have characteristic C1: G72 base pairs [Marck et al., RNA, Vol. 8, 2002, p. 1 189-1232 (ref. 21)], anticodons, and A73 identities It has a determinant (Refs.
  • eubacteria have the same anticodon, A73, but the opposite G1: C72 base pair and a characteristic long variable arm (Reference 21) as identity determinants [Himeno et al., Nucleic Acids Research. Vol. 18, 1990, p. 68 15 -68 19 (Reference 22)] (Fig. 1). Reversing the 1:72 base pair of eubacterial and eukaryotic tRNA Tyf to C1: G72 and G1: C72, respectively, reverses species-specific recognition by TyrRS, regardless of the length of the variable arm [ Wakasugi et al., The EMBO Journal Vol. 17, 1998, p. 297-305 (Reference 23)]. Therefore, both the eubacteria type and the bacterium / eukaryote type TyrRS need to recognize 1:72 base pairs of tRNA. It has been suggested that
  • TyrRS from archaebacteria and eukaryotes are similar to each other, but their TyrRS and TyrRS of eubacteria are low in similarity (Fig. 2).
  • Fig. 2 For example, when sequence homology is analyzed using the PSI-BLAST program (ht tp: // www. Ncbi. Nlm. Nih. Gov / blast /), M.zo3 ?? 59% similarity was found between / and human TyrRS, whereas the e-value was high at 0.22 between M. jannaschii and B. Wear No gender is found.
  • the present invention relates to a crystal of a triple complex of TyrRSZtRNA ⁇ / L-tyrosine of M.a / ?? 7 '/, TyrRS having a tyrosine binding pocket, and 3-aodotyrosine as a substrate rather than aaRS activity using tyrosine as a substrate.
  • the present invention provides the following crystals.
  • the present invention provides a TyrRS having the following tyrosine binding pocket.
  • the present invention provides the following mutant TyrRS and a method for producing the same based on the information on the tyrosine binding pocket structure of (3) above.
  • amino acid sequence represented by SEQ ID NO: 1 a sequence in which one or more amino acid residues of ⁇ Tyr32, His70, Aspi58 '' are replaced with another amino acid residue, or It consists of a sequence in which at least one amino acid residue of “Tyr 32, Aspl 58, Hisl 77” has been replaced with another amino acid residue, and has a 3-terminal activity lower than the aaRS activity using tyrosine as a substrate. Mutant TyrRS characterized by enhanced aaRS activity using eodotyrosine as a substrate.
  • mutant TyrRS of (4) the sequence in which ⁇ Tyr32, His70, Aspl58 '' is replaced with ⁇ Tyr32, Ala70, Thrl58 '', or ⁇ Tyr32
  • a mutant TyrRS comprising a sequence in which “His 70, As pl 58” is replaced with “Thr 32, Thr 70, G lul 58”.
  • (C) A method comprising selecting, from a mutant TyrRS library, one having enhanced aaRS activity using a target tyrosine derivative as a substrate over aaRS activity using tyrosine as a substrate.
  • the amino acid residue selected in the step (A) is a combination of Tyr32, His70, and Asp158, or a combination of Tyr32, Aspl58, and Is177.
  • a method for producing a mutant TyrRS comprising:
  • the present invention provides a TyrRS having the following anticodon G34 binding pocket.
  • the present invention provides the following mutant TyrRS and a method for producing the same based on information obtained from the anticodon G34 binding pocket structure of (11).
  • the present invention provides the following mutant TyrRS and a method for producing the same.
  • the aaRS activity using 3-odotyrosine as a substrate is higher than the aaRS activity using tyrosine as a substrate, and the aminoacylation of amber repressor tRNA is higher than that of TyrRS consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • Mutant TyrRS characterized by increased reaction rate.
  • the amino acid sequence of the mutated TyrRS of (17) comprises one or more amino acids deleted, substituted, or added,
  • the aaRS activity using 3-odotyrosine as a substrate is higher than the aaRS activity using tyrosine as a substrate, and the rate of aminoacylation of ambassador-NA is higher than that of TyrRS consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • TyrRS consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • the present invention provides the following kit for producing a polypeptide incorporating a tyrosine derivative and a method for producing a polypeptide.
  • a kit for producing a polypeptide incorporating a tyrosine derivative comprising:
  • a method for producing a polypeptide characterized in that:
  • Figure 1 shows a comparison of the sequences of tRNA Tyr and TyrRS, showing the secondary structure of the tRNA Tyr species.
  • FIG. The M. zoa /? ⁇ / 'And yeast tRNA sequences are shown as transcripts.
  • the letter and the boxed area indicated by the arrow indicate the same identity element for each tRNA T.
  • FIG. 2 shows a sequence alignment of TyrRS.
  • the sequence is jannaschii, Archaeoglobus fulgidus, human, yeast (Itotsuki moon capsule) and zozo stearotlwr hi 1 lus, Thermus thermophi lus ⁇ M. JannascJii itT. Each is shown above and below the alignment.
  • the coil is a "helix” and the arrow is] 3 strands. Letters indicate conserved residues. Alignment was performed with CLUSTALX (Thompson et al., Nucleic Acids Research, Vol. 25, pp. 4876-4882, 1997) and corrected for each other based on the secondary structure of M. jannaschii and T./force //.
  • FIG. 3 is a diagram showing the structure of plasmid pMYR-Lac (Am).
  • M J YR 1 DNA fragment containing M. zo a / was / '/ suppressor NA gene.
  • PEYR Fragment containing expression promoter overnight.
  • LacZ (amb) DNA fragment containing a-lac (Am).
  • FIG. 4 shows the tyrosine binding pocket of yavM ⁇ 7 TyrRS. Black represents ⁇ , and diagonal lines represent N.
  • FIG. 5 shows the G34 binding pocket of the tRNA Tyr anticodon of zoa / M / 'TyrRS. Black represents ⁇ , and diagonal lines represent N.
  • FIG. 6 is a lipon model stereogram showing the structure of the ./??7 TyrRS / tRNA Tyr / L-tyrosine complex.
  • Two tyrosine molecules are represented by the CPK model.
  • Residues 203-209 and the 3 'terminal CCA chain of the tRNA T molecule are not shown because they are disordered.
  • FIG. 7 is a stereogram of the molecular structure of one TyrRS subunit. Only C position is shown. Each 20th residue is indicated by a bullet.
  • Figure 8 is a stereo view of the I FO-F c I simulated annealing omit electron density map (3.0 ⁇ ) around the tyrosine binding pocket at 1.95 ⁇ (1.95 ⁇ 1 ( ⁇ 1 ⁇ ) resolution). .
  • Figures 9 ⁇ and 9 9 compare the overall structure of TyrRS ⁇ tRNA.
  • Fig. 9A shows a stereogram in which M. yaw? 'Is superimposed on humans
  • Fig. 9B shows M. jannaschiit. It is a stereo view in which T.//7'/hi 5 is superimposed.
  • FIG. 10 is a diagram showing the] 3-310-iS motif inserted into the C-terminal domain of I.zoa / H? / TyrRS.
  • FIG. 11 is a diagram showing the superposition of what is equivalent to FIG. 9B viewed from the double axis.
  • FIG. 12 is a diagram showing the recognition of the exceptor stem of TyrRS, and is a stereogram of the exceptor-stem binding site of M. jannaschii TyrRS.
  • Figure 13 is a schematic ( stereogram ) around the first base pair of the tRNA Tyf of the M.zo a / w? C force / complex. Since the tRNA was generated by a lipozyme self-cleavage reaction, there is no phosphate at the 5 'end of the tRNA. Nucleotides 1, 72, and 73 of each tRNA are shown in the stick model. Hydrogen bonds and other interactions are indicated by dashed lines. The Ripon model shows the N-terminal region T, the Rosmanfold domain, and the CP1 domain.
  • FIG. 14 is the corresponding diagram in T. 77zw. Nucleotides 1, 72, 73 of each tRNA Tyr are shown in the stick model. Hydrogen bonds and other interactions are indicated by dashed lines.
  • the Ripon model shows the N-terminal region T, Rosmanfold domain, and CP1 domain.
  • FIG. 15 shows the anticodon recognition of TyrRS, and is a stereogram showing anticodon recognition by .ya / w? D // TyrRS. Hydrogen bonds are indicated by dashed lines. Anti-codon triplets are represented by a stick model. The C-terminal domain is shown in a Ripon model.
  • Figure 16 is a correspondence diagram (stereo diagram) of the T./ ⁇ / complex. Hydrogen bonds are indicated by dashed lines. Anticodon triplets are represented by a stick model. The C-terminal domain is shown in the Ripon model.
  • FIG. 17 is a graph showing a comparison of the initial speed of aminoacylation between the wild type and several mutants.
  • FIGS. 18A and 18B show the amino acid residues of TyrRS that recognize the side chain of L-tyrosine (stereo diagram).
  • FIG. // Shows the amino acid binding site of the triple complex
  • FIG. 18B is .st ea r 01 he rnwph i 1 us, i.
  • Two residues of M. jannaschii, Glul07 and Leul62, are each ew of B. stearothe rmoph i 1 us Corresponding to
  • Figure 19 is a photograph of a crystal of a native (left) and selenomethionine-labeled (right) TyrRS-tRNA Tyr -tyrosine complex.
  • FIG. 20 is a diffraction image of a native crystal.
  • the arrow corresponds to 1.95 ⁇ (1.95 ⁇ 10-1 ⁇ 2 ⁇ ) resolution.
  • FIG. 21 shows an XAFS spectrum of Se atom of the selenomethionine-labeled complex crystal. The measurement of the diffraction data was performed at the peak wavelength indicated by the arrow.
  • Figure 22 is the Fourier diagram of the extraordinary dispersion difference (4 cut-off) of the selenomethionine-labeled complex.
  • the selenomethionine side chain is shown by a stick model, and the main chain of TyrRS is shown by a ribbon model.
  • FIG. 23 shows the crystal packing (stereo diagram) of the TyrRS ′ tRNA Tyt> complex. In the center, you can see a dimerized molecule with crystallographic symmetry.
  • FIG. 24 shows non-natural amino acids specifically recognized by the TyrRS mutant of j'azwasdz / i, and shows only the side chains.
  • FIG. 25 shows the amino acid sequence of janmschii. * Indicates the end of translation.
  • FIG. 26 is a photograph of an SDS-PAGE gel in which the expression of GFPuv (ajnber) was examined.
  • FIG. 27 is a photograph of a gel of SDS-PAGE in which the expression of TT1865 was examined.
  • a tyrosine derivative refers to one in which an arbitrary substituent is introduced into any of atoms constituting tyrosine, and there is no limitation on the position where the substituent is introduced. Specifically, for example, a thioxacin derivative having a side chain shown in FIG. 24 can be mentioned.
  • the TyrRS mutant of the present invention has a mutation at a specific position in the amino acid sequence, and as long as the desired activity is maintained, one or several amino acids are deleted at amino acid residues at other positions, Substitutions or additions are also included.
  • the TyrRS mutant of the present invention has a mutation at a specific position in the amino acid sequence, and as long as the desired activity is maintained, 70% or more of amino acid residues at other positions Those having homology, preferably 80% or more homology, more preferably 90% or more homology are included.
  • the present invention provides the atomic structure coordinates of a ternary complex as determined by high resolution three-dimensional structure and X-ray crystallography. Specific methods for crystallization of the triple complex and X-ray analysis of its structural coordinates are as described in Examples.
  • the crystal of the triple complex of TyrRS of jannaschii and tRNA T of ya / w? / '/' And L-tyrosine (hereinafter referred to as triple complex of TyrRSZtRNA ⁇ ZL-tyrosine) of the present invention has a space group of 1 Yes, the unit cell is (15.6 nm).
  • the unit cell refers to the smallest simple volume element of the crystal, and the space group refers to the symmetry of the unit cell.
  • Table 3 shows the atomic structure coordinates of the triple complex of TyrRSZtRNA ⁇ ZL _ tyrosine of the present invention obtained at a resolution of 1.95 A ( ⁇ ⁇ ⁇ -' ⁇ ).
  • atomic numbers 1 to 2415 correspond to TyrRS
  • 2416 to 2428 correspond to L-tyrosine
  • 2429 to 4006 correspond to tRNA
  • 4007 to 4374 correspond to trapped water.
  • the atomic structure coordinates of the triple complex of TyrRSZtRNA T "ZL-tyrosine mean those which match or substantially match the coordinates shown in Table 3, and more specifically, those shown in Table 3.
  • the coordinates shown when they are superimposed using backbone atoms (N, Co !, C and O), they are less than about 1.5 A (0.15 nm), preferably about 1.0 A (0.1 nm).
  • the atomic coordinates shown in Table 3 reflect the tertiary structure of TyrRS of. Jannaschii bound to tRNA Tyf and L-tyrosine and provide much useful information. In particular, it can be used to specify the position of amino acid substitution for TyrRS modification of archaebacteria. In addition, as described above, the similarity of TyrRS between archaea and eukaryotic cells suggests that it can be used to identify the position of amino acid substitutions for eukaryotic TyrRS modification shown in Table 3. Conceivable.
  • the tyrosine-binding pocket structure of TyrRS revealed in the present invention has a structure defined by atomic numbers 1 to 24 15 in atomic coordinates shown in Table 3.
  • Amino acid residues Ty r 32 (atomic numbers 259 to 270), I 1 e 33 (atomic numbers 27 1 to 278), Gly 34 (atomic numbers 279 to 282), Ph e 35 (atomic numbers 283 to 293), G lu 36 (atomic numbers 294-302), Le eu 65 (atomic numbers 525-532), A1a67 (atomic numbers 541-545), His 70 (atomic numbers 562-571), Tyr 1 51 ( Atomic numbers 1230 to 1241), G1n155 (atomic numbers 1265 to 1273), Asp158 (atomic numbers 1289 to 1296), G1n173 (atomic numbers 1398 to 1406), and His 177 (Atomic numbers 1435 to 1444).
  • FIG. 4 shows an enlarged view of this tyrosine binding pocket. In Fig. 4, the dotted line indicates a hydrogen bond.
  • the L-tyrosine amino and haponyl groups of the substrate form a hydrogen bonding network at G1n173, Tyr151, and G1n155. That is, the amino group of L-tyrosine of the substrate forms a hydrogen bond with the carbonyl group of Glnl 73, the hydroxyl group of Tyr151 and the carbonyl group of G1n155, respectively, and The tyrosine radical group forms a hydrogen bond with the amino group at G1n173, and further forms a hydrogen bond with the carbonyl group at G1n173 and the amino group at G1n155.
  • the aromatic ring of tyrosine is recognized by the side chains of Leu 65, His 70, and G1n155, and also by the main chains of Ile33, Gly34, and Phe35. (Behind tyrosine, not shown in Figure 4).
  • the hydroxyl group on the aromatic ring of tyrosine is recognized by hydrogen bonding at Tyr32 and Aspl58 inside the pocket. It is.
  • the water molecule is trapped in hydrogen bonds by His177 and Tyr32 and is also proximal to the tyrosine side chain.
  • This tyrosine binding pocket is thought to determine the substrate specificity of wild-type TyrRS for tyrosine. Therefore, in order to modify the substrate specificity of TyrRS for tyrosine, it is considered effective to mutate the amino acid residues constituting the tyrosine binding pocket.
  • the present invention provides a mutant TyrRS in which aaRS activity using a desired tyrosine derivative as a substrate is higher than aaRS activity using tyrosine as a substrate, based on the information on the tyrosine binding pocket structure (hereinafter simply referred to as the substrate specificity of the present invention). (Also referred to as mutant TyrRS). That is,
  • (C) a method comprising selecting, from a mutant TyrRS library, one in which aaRS activity using a target tyrosine derivative as a substrate is higher than aaRS activity using tyrosine as a substrate.
  • a mutant TyrRS in which aaRS activity using a target tyrosine derivative as a substrate is higher than aaRS activity using tyrosine as a substrate is a mutant TyrRS in which the substrate affinity for a desired tyrosine derivative is higher than that for tyrosine.
  • Tyrosin also has an increased substrate affinity for the desired tyrosine derivative, which means that the activity value (the reaction rate cat divided by the Michaelis constant K m ) for the target tyrosine derivative is greater than the activity value for tyrosine.
  • the activity value can be measured by an in vitro assay, or the relative magnitude of the activity value can be determined from genetic data.
  • the atom into which the substituent is introduced may be any of the carbons constituting the aromatic ring of tyrosine.
  • the carbon atom at the 2- or 3-position of the tyrosine aromatic ring If you want to introduce a substituent into the tyrosine, there are two places due to the line symmetry of tyrosine.
  • a tyrosine derivative in which the hydroxyl group at position 4 of the tyrosine aromatic ring is replaced with another substituent may be used.
  • mutants having substrate specificity for various tyrosine derivatives as shown in FIG. 24 have already been obtained, various substrate specificities can be obtained from the mutation of M.zo aim 7 '. It has been demonstrated that it can have.
  • the site of mutation can be specified more rationally, so that it is possible to obtain a mutant with higher substrate specificity for a tyrosine derivative that is already known to be a substrate.
  • the possibility of obtaining mutants having substrate specificity for thioxacin derivatives having new positions and types of substituents has been opened.
  • step (B) a mutant TyrRS library in which the selected amino acid residue has been replaced with another amino acid residue is prepared.
  • random substitution of amino acid residues can be performed by a known gene manipulation technique using PCR or the like.
  • the 3-substituted two kinds if you select three amino acid residues, the combination of amino acid substitutions should be viewed attempt is by two ways 0 3, 1 6 0 0 0 ways to suit.
  • a library of mutant genes in which the nucleotide residues corresponding to these three positions on the gene (9 residues each in total) have been changed to a random sequence can be created using PCR.
  • the expression of the tyrosine (+) tyrosine derivative (-) in the medium of the tyrosine (+) tyrosine derivative (-) does not occur, but the tyrosine (+) tyrosine derivative ( +) In the medium May be selected to cause an amber sub-resolution.
  • a strain in which a member mutation has been introduced into Lac is transformed with a library of mutant genes in which a random sequence has been changed in combination with an amber suppressor tRNA, and a tyrosine (+) tyrosine derivative (- In the medium of (1), amber-subversion (Lac-) does not occur.
  • an amber-subversion (Lac +) can be selected.
  • the target one can be easily selected from many transgenic cells.
  • an example of a method for modifying the amino acid specificity of jan schii TyrRS using the method for producing a mutant TyrRS of the present invention will be specifically described.
  • the first and third underlines indicate the site and the dl II site.
  • the second underline indicates the structural gene region of the M. jannas chii suppressor 1-tRNA.
  • DNA fragment containing ⁇ -lac (Am) (SEQ ID NO: 7)
  • the tyrosine ring in the three-dimensional structure of the L-tyrosine binding site of TyrRS (Fig. 4) Select the next 3 residues located near the 3rd position (2 left and right places).
  • N A gene substituted with any of A, G, C, T, ⁇ : 6 or 1 is prepared by the following two-step PCR and expressed in E. coli by the method described in [2].
  • Step 1 of PCR Including the following four rounds of PCR using primers 1-18.
  • PCR.1 Use primer 1 (SEQ ID NO: 3) and primer 3 (SEQ ID NO: 8).
  • PCR.2 Use Primer 4 (SEQ ID NO: 9) and Primer 5 (SEQ ID NO: 10).
  • PCR.3 Primer 1 (SEQ ID NO: 11) and Primer 1 (SEQ ID NO: 12) are used.
  • PCR.4 Use primer 8 (SEQ ID NO: 13) and primer 2 (SEQ ID NO: 4).
  • the reaction conditions may be as follows.
  • Step 2 of PCR Mix 10 nanograms of each PCR product obtained in PCR.1 to 4 and use primers 1 and 2 as type III.
  • PCR.1 Use primer 1 (SEQ ID NO: 3) and primer 1 (SEQ ID NO: 8).
  • PCR.2 Use primer 4 (SEQ ID NO: 9) and primer 6 (SEQ ID NO: 11).
  • PCR.3 Use primer 8 (SEQ ID NO: 13) and primer 1 (SEQ ID NO: 14).
  • PCR.4 Use primer 10 (SEQ ID NO: 15) and Primer 1 (SEQ ID NO: 4).
  • the mutant TyrRS expression plasmid prepared in [5] or [6] is introduced into the MV1184 * strain by the transformation method, respectively, and plated on an LB * plate. Collect the colonies (8000 or more) that have formed after the incubation at 37 ° C for 18 hours or more, inoculate them again on LB «(IY) plates, observe the colonies again after keeping the incubation for 24 hours or more.
  • Each colony contains one mutant TyrRS gene clone. If the mutant TyrRS has the activity to bind L-tyrosine (or IY or L-tyrosine if IY is added) in the plate to Mjsup-tRNA, then the mutant in the G-lac (Am) gene Substitution of the mutation results in a blue colored colony. Therefore, the blue-stained colonies on the LB «(IY) plate may contain the mutant TyrRS gene that has the activity to bind IY or L-tyrosine to M] 'sup-tRNM. Understand. Therefore, transfer one blue-stained colony to each plate and LB (IY) plate, incubate at 37 ° C for 24 hours or more, and observe coloring again.
  • the colony which is blue on the LB ⁇ ( ⁇ ) plate and white on the LB plate, contains a mutant TyrRS gene that has the activity to bind only ⁇ to Mjsup-tRNA. Therefore, the mutant TyrRS gene is recovered from each colony.
  • composition of each plate can be as follows.
  • LB * plate LB plate containing 100 milligrams of ampicillin and 25 milligrams of chloramphenicol per liter.
  • LB plate 1 mM (final concentration) of isopropyl-tothio-D-galactopyranoside per liter, 40 mg of 5-bromo-4-chloro-3-indolyl;; 40 mg of 3-D-galactopyranoside, IY LB plate containing 0.1 g.
  • LB (IY) plate 1 mM (final concentration) of isopropyl-1-thio-D-galactovyranoside per 1 liter, 40 mg of 5-promo-4-octanol-3-indolyl- ⁇ -D-galactopyranoside, ⁇ LB plate containing 0.1 g.
  • the mutant thus obtained is a mutant TyrRS having a higher affinity for a desired tyrosine derivative than tyrosine. That is,
  • a mutant TyrRS produced by a method comprising selecting a substance having an enhanced aaRS activity using a target tyrosine derivative as a substrate rather than an aaRS activity using tyrosine as a substrate, for example,
  • a target tyrosine derivative as a substrate
  • tyrosine as a substrate
  • the desired amber-modified gene By expressing the desired amber-modified gene at a desired position in combination with an amber repressor tRNA derived from an archaebacteria or a eukaryote by the method described below, the desired tyrosine can be placed at a desired position. It is preferably used for production of a polypeptide into which is incorporated.
  • amino acid sequence represented by SEQ ID NO: 1 (amino acid sequence of wild-type TyrRS of .zo //; 70, a sequence in which one or more amino acid residues of Asp 158 are replaced with another amino acid residue, or one or more amino acid residues of Tyr32, Aspl 58, and His 177
  • a mutant TyrRS comprising a sequence substituted with an amino acid residue, characterized in that aaRS activity using a 3-substituted tyrosine as a substrate is higher than aaRS activity using a tyrosine as a substrate, can be obtained by, for example, a method described below.
  • a desired gene having an amber mutation at a desired position is expressed, thereby incorporating the target 3-substituted tyrosine at a desired position.
  • a desired gene having an amber mutation at a desired position is expressed, thereby incorporating the target 3-substituted tyrosine at a desired position.
  • mutant TyrRS of the present invention in the amino acid sequence represented by SEQ ID NO: 1, one or more amino acid residues of Tyr32, His70, and As158 are replaced with another amino acid residue. Or a sequence in which one or more amino acid residues of Tyr32, Aspl58, and His177 have been substituted with another amino acid residue, and based on aaRS activity using tyrosine as a substrate. Is also a mutant Ty rRS characterized by enhanced aaRS activity using a 3-substituted tyrosine as a substrate.
  • amino acid sequence represented by SEQ ID NO: 1 a sequence in which one or more amino acid residues of Tyr32, His70, and Asp158 have been substituted with another amino acid residue, or Tyr32, It consists of a sequence in which one or more amino acid residues of Asp 158 and His 177 are replaced with another amino acid residue, and has aaRS activity using 3-odotyrosine as a substrate higher than aaRS activity using tyrosine as a substrate
  • the mutation TyrRS which is characterized in that it has been performed, is combined with an archebacterial or eukaryotic member-subtractor tRNA, for example, by the method described below, and is supposed to have an amber mutation at a desired position.
  • the gene By expressing the gene, it is preferably used for the production of a polypeptide incorporating a 3-substituted tyrosine, preferably a 3-halogenated tyrosine, particularly 3-ododotyrosine at a desired position.
  • a mutant TyrRS of the present invention in the amino acid sequence represented by SEQ ID NO: 1, one or more amino acid residues of Tyr32, His70, and As158 are replaced with another amino acid residue. Or a sequence in which at least one amino acid residue of Tyr32, Aspl58, and His177 has been substituted with another amino acid residue, and based on the aaRS activity using tyrosine as a substrate.
  • T yr RS characterized by enhanced aaRS activity using 3-hydroxytyrosine as a substrate.
  • amino acid sequence represented by SEQ ID NO: 1 a sequence in which His 70 is substituted with A 1 a and Asp 158 is substituted with Thr (Ty r 32—A la 70—Th r 158), or Mutation consisting of a sequence in which Tyr32 is replaced by Thr, His70 is replaced by Thr, and Asp158 is replaced by G1u (Thr32—Thr70—G1u158) Provide TyrRS.
  • this mutant has high specificity for 3-odotyrosine is that in the selection method described above, amber suppression (Lac-) does not occur in the medium of tyrosine (+) 3 -odotyrosine (-). However, tyrosine (+) has been demonstrated by the occurrence of amber-substitution (Lac +) in the medium of 3-hydroxytyrosine (+).
  • mutant TyrRS of the present invention in the amino acid sequence represented by SEQ ID NO: 1, a sequence in which His 70 is replaced by A1a and Asp 158 is replaced by Thr (Ty r 32-A la 70-Th rl 58), or a sequence in which Ty r 32 is replaced with Thr, His 70 is replaced with T hr, and Asp 158 is replaced with G 1 u (T hr 32- It is a mutant TyrRS containing Th r 70 -G 1 u 158).
  • 3-Halogenated tyrosine such as 3-hydroxytyrosine and 3-bromotyrosine
  • the mutated TyrRS of the present invention having enhanced substrate specificity for 3-halogenated tyrosine can be used for the production of an aroprotein incorporating a 3-monohalogenated tyrosine, and such an aroprotein may be a protein.
  • Function ⁇ It is useful as a material for structural analysis and may also be a target for drug discovery.
  • the anticodon G34 binding pocket structure of the present invention thus identified has the structure defined by atomic numbers 1 to 2415 in atomic coordinates shown in Table 3.
  • the amino acid residue P It is an anticodon G 34 binding pocket structure formed by he 261, His 283, Pro 284, Met 285, and Asp 286.
  • This anticodon G34 binding pocket is shown in FIG.
  • the base portion of G34 stacks between the rings of Phe261 and His283, and the nitrogen atom at position 1 and the amino group at position 2 are both recognized by hydrogen bonding with Asp286. I have.
  • the present inventors have developed an aminoacylate tRNA, a G34C variant of tRNA ⁇ (a tyrosine anticodon GUA in which the first letter G of the GUA has been replaced with a C and the anticodone has become a CUA). To increase the efficiency of the conversion, a mutation was introduced at residue 286, and its effectiveness was confirmed.
  • one embodiment of the present invention relates to an aminoacylation reaction for Amber Sublesser-tRNA having a sequence in which Asp 286 is replaced with another amino acid residue in the amino acid sequence shown in SEQ ID NO: 1.
  • This is a mutant TyrRS derived from zo '3 / ?; 7.
  • any of known methods may be used.
  • a primer in which the nucleotide sequence encoding the position of the target amino acid has been replaced with a nucleotide sequence encoding the amino acid to be modified Amplifying the DNA substituted with the nucleotide sequence encoding the amino acid to be modified, joining the amplified DNA fragments to obtain DNA encoding the full-length a aRS mutant, It can be easily produced by expressing it using a host cell such as Escherichia coli.
  • the primer used in this method has 20 to 70 bases, preferably about 20 to 50 bases. This primer has 1 to 3 base mistakes with the original base sequence before modification. It is preferable to use a relatively long one, for example, one having 20 bases or more, because it will result in a match.
  • the present inventors have found that, in this Asp286 substitution, when cytosine comes to the 34th base of NA in wild-type TyrRS, even if it comes to the position where the base is inverted like G34, Asp286 and base Asp286 was replaced with larger side chains, Glu, Phe, Ile, Leu, Gin, Arg, and Tyr, taking into account the possibility that satisfactory interaction might not be obtained due to the distance of A mutant was prepared.
  • the present invention provides a mutant TyrRS characterized by having an increased aminoacylation reaction rate for a member-subpressor tRNA as compared to a TyrRS comprising an amino acid sequence.
  • Asp 286 is substituted with Gln, Arg, or Tyr.
  • “high reaction rate” means that the initial rate is high when the substrate concentration and the enzyme concentration are kept constant.
  • either the Michaelis constant K m for that substrate may be lower, or the reaction rate constant Keal may be higher. That is, a mutant TyrRS having an increased aminoacylation reaction rate to amber suppressor tRNA as compared with TyrRS (wild type) consisting of the amino acid sequence represented by SEQ ID NO: 1 was obtained by using an amber suppressor tRNA as a substrate.
  • Michaelis constant K m of the mutant or lower than wild type when, or reaction rate constant K eat mutants means higher than the wild type.
  • This mutant TyrRS is preferably used for polypeptide production by expressing a nucleic acid having an amber mutation introduced at an arbitrary position in combination with an amber suppressor tRNA derived from an archaebacteria or a eukaryote.
  • mutant TyrRS of the present invention the amino acid sequence represented by SEQ ID NO: 1
  • a mutant TyrRS containing a sequence in which Asp286 is substituted with another amino acid residue has a higher aminoacylation rate for the amber suppressor tRNA than a TyrRS containing the amino acid sequence represented by SEQ ID NO: 1.
  • Mutant TyrRS characterized by enhanced levels.
  • the present invention further relates to a mutant TyrRS in which aaRS activity based on a tyrosine derivative is higher than aaRS activity using tyrosine as a substrate, wherein an amino acid residue corresponding to Asp 286 in SEQ ID NO: 1 is G 1 n
  • a mutant TyrRS having an increased aminoacylation reaction rate with respect to Amber Sublesser-tRNA, wherein the method comprises substitution with Tyr, Arg, or Tyr.
  • a mutant TyrRS in which aaRS activity using a tyrosine derivative as a substrate is higher than aaRS activity using tyrosine as a substrate is one or more of the above-mentioned ⁇ Tyr32, His70, Asp158 '' It consists of a sequence in which an amino acid residue is replaced with another amino acid residue, or a sequence in which one or more amino acid residues of “Tyr32, Aspl58, Hisl77” are replaced with another amino acid residue
  • mutant TyrRS having improved substrate specificity for tyrosine derivatives described in the above-mentioned references 4, 11, 12 and the like can be mentioned.
  • the method of the present invention is based on these substrate-specific variants by further substituting Asp286 or an amino acid residue corresponding thereto with another amino acid residue.
  • Asp286 or an amino acid residue corresponding thereto can be easily determined by determining the amino acid sequence of each mutant by a well-known method and comparing it with the amino acid sequence of SEQ ID NO: 1.
  • This mutant TyrRS has a higher aminoacylation rate for the amber-subpressor tRNA than the TyrRS before the substitution of Asp286, so it can be combined with an amber-subpressor tRNA derived from archaebacteria or eukaryotes.
  • a nucleic acid having an amber mutation introduced at an arbitrary position it is preferably used for producing a polypeptide having a tyrosine derivative incorporated at an arbitrary position.
  • the aaRS activity using 3-odotyrosine as a substrate is higher than the aaRS activity using tyrosine as a substrate, and it is more effective against Amber Sublessa-NA than TyrRS consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • the present invention further relates to the amino acid sequence represented by SEQ ID NO: 1, in which His 70 is replaced with A 1 a and Asp 158 is replaced with Thr (Ty r 32 -A 1 a 70 -Th r 158) and a sequence in which A sp 286 is substituted with G 1 n, Ar g, or Tyr, or Ty r 32 is substituted with Thr, His 70 is substituted with Thr, and A sp 158 is G 1 consists of a sequence substituted with u (Thr32—Thr70—Glul58) and Asp286 substituted with Gln, Arg, or Tyr, rather than aaRS activity using tyrosine as a substrate.
  • aaRS activity using 3-odotyrosine as a substrate is enhanced, and the aminoacylation rate for amba-sublesser-tRNA is increased as compared to TyrRS consisting of the amino acid sequence represented by SEQ ID NO: 1. Mutant TyrRS is also provided.
  • This mutant TyrRS is also used in combination with an archaebacterial or eukaryotic amber-subtractor tRNA to express a nucleic acid having an amber mutation introduced at an arbitrary position, whereby a 3-position-substituted cytosine synthase can be obtained at an arbitrary position. Particularly, it is preferably used for production of a polypeptide incorporating 3-odotyrosine.
  • mutant TyrRS of the present invention in the amino acid sequence represented by SEQ ID NO: 1, His 70 is replaced with A1a, Asp 158 is replaced with Thr (Ty r32- A1a70-Thr158) and a sequence in which Asp286 is substituted with G1n, Arg, or Tyr, or Tyr32 is substituted with Thr, and His70 is substituted with Thr.
  • a sequence in which Asp158 is substituted with G1u (Thr32—Thr70—G1u158) and Asp286 is substituted with G1n, Arg, or Tyr, and tyrosine is included.
  • the aaRS activity using 3-odotyrosine as a substrate is higher than the aaRS activity using as a substrate, and the aminoacylation rate for amber repressor tRNA is higher than that of TyrRS consisting of the amino acid sequence represented by SEQ ID NO: 1. It is a mutant TyrRS characterized by an enhancement. (8) Polypeptide production and purification
  • the mutant TyrRS thus obtained can be used in combination with an archaeal or eukaryotic sub-tRNA to produce a polypeptide incorporating the tyrosine derivative in vivo or in vivo.
  • a desired polypeptide is synthesized by a polypeptide synthesis system using the mutant TyrRS and an archaebacteria- or eukaryotic-derived tRNA capable of binding to a tyrosine derivative in the presence of the mutant TyrRS. It is intended to provide a method for producing a polypeptide incorporating a cytosine derivative, which comprises expressing a polypeptide containing an unnatural amino acid using a desired gene having a nonsense mutation at a position.
  • any synthesis system can be used as long as it is an expression system that can be expressed using the above-mentioned mutant TyrRS, a bindable sublesser tRNA, and a desired gene.
  • the “cell-free polypeptide synthesis system” means a system for synthesizing polypeptide in vitro using a cell extract, and reads the mRNA information of mRNA and reads it on the liposome.
  • a cell-free translation system for synthesizing polypeptides and a system containing both a cell-free transcription system and a cell-free translation system for synthesizing RNA using DNA as type II.
  • the requirements for cell-free polypeptide synthesis systems include:
  • Desired cell extract preferably prokaryotic bacterial extract
  • the cell extract of (1) contains ribosomes, tHNA, enzymes necessary for polypeptide synthesis, etc., and is a concentrated cell extract of Escherichia coli having high polypeptide synthesis activity, particularly Escherichia coli A concentrated S30 cell extract can be used.
  • the concentrated cell extract can be obtained by concentrating the above crude cell extract by a concentration method such as dialysis, ultrafiltration, or polyethylene glycol (PEG) precipitation.
  • the concentration of E. coli S30 cell extract can be concentrated by a known method (Zubay et al. (1973) Ann. Re. Genet. 7: E. coli A19 (rna, met)) in a closed system with shaking or stirring.
  • E. coli S30 extract (also available from Promega) obtained from E. coli is used as the inner dialysis solution, and dialyzed against the outer dialysis solution through a dialysis membrane with a molecular weight limit of 1 000 to 14000.
  • the dialysis external solution is composed of a buffer solution containing potassium acetate, magnesium acetate, and dithiothreitol, and a polyethylene glycol or sucrose Zepichlorhydrin water-soluble synthetic copolymer (eg, SI GMA Ficoi l).
  • the cell extract derived from Escherichia coli is preferably concentrated, but may not be concentrated.
  • the term “enriched cell extract” refers to a crude extract of eukaryotic and prokaryotic cells containing components required for polypeptide synthesis such as ribosomes and tRNA, dialysis, ultrafiltration, and precipitation (3). Nakano et al., Journal of Biotechnology, 46 (1996) 275-282), etc., or those enriched by a newly discovered enrichment method, wherein the extract is involved in the in vivo synthesis of polypeptides. And translation / translation / translation system components. “Enrichment” means an increase in the concentration of the total protein in the extract as an index.
  • the cell extract contains polyribosomes, tRNA, etc. Contains components required for peptide synthesis.
  • the method described in Pratt, JM et al., Transcription and translation-a practical approach, (1984), pp. 179-209, Henes, BD and Higgins, SJ, IRL Press, Oxford) is used. it can. Specifically, crushing by French press (Pratt et al., Supra) and crushing using glass beads (Kim et al., Supra) Can be done by
  • a preferred cell extract is E. coli S30 cell extract.
  • the S30 cell extract can be prepared from Escherichia coli A19 (rna, met) according to known methods, for example, the method of Pratt et al. (Supra), or can be prepared from Promega or Novagen. A commercially available product may be used.
  • the cell extract needs to be concentrated so as to increase its total protein concentration.
  • Concentration can be performed by any means such as ultrafiltration (including ultrafiltration centrifugation), dialysis, and PEG. It can be performed by precipitation or the like.
  • the degree of concentration is usually 1.5 times or more, preferably 2 times or more.
  • a cell extract derived from Escherichia coli it can be concentrated to 1.5 to 7 times or more by ultrafiltration centrifugation and 1.5 to 5 times or more by PEG precipitation, but if it exceeds 4 times, handling becomes difficult.
  • PEG precipitation Nakano, H. et al., Supra.
  • a polypeptide and a nucleic acid are precipitated and mixed by mixing an aqueous PEG solution with a cell extract, and a concentrated cell extract can be obtained by dissolving this in a small amount of buffer.
  • Concentration by dialysis is performed, for example, by using a cell extract as a dialysis solution in a closed system that can be shaken or stirred, and dialyzing the dialysis solution through a dialysis membrane (for example, with a molecular weight limit of 1000 to 1400). Obtainable.
  • the outer dialysis solution is a buffer solution containing potassium acetate, magnesium acetate, and dithiothreitol, a PEG (eg, # 8000), a sucrose Z-epiclorhydrin water-soluble synthetic copolymer (eg, SI GMA And a polymer absorbent such as Fico 11 1).
  • Polymer absorbents are essential for absorbing moisture.
  • Cell-free polypeptide synthesis systems include concentrated cell extracts such as Escherichia coli S30, ATP (adenosine 5, monotriphosphate), GTP (guanosine 5, 1 Triphosphate), CTP (cytidine 5, monotriphosphate), UTP (peridine 5, monotriphosphate), buffers, salts, amino acids, RNase inhibitors, antimicrobial agents, RNA polymerase if necessary (When DNA is used as type I) and tRNA.
  • concentration of the additive component can be arbitrarily selected.
  • a buffer such as Hepes-K ⁇ H or Tris-OAc can be used.
  • salts are acetate (eg, ammonium salt, magnesium salt, etc.), glutamate, etc.
  • antibacterial agents are sodium azide, ampicillin, etc.
  • Amino acids are the 20 amino acids that make up the polypeptide.
  • RNA polymerase is added to the reaction system.
  • a commercially available enzyme such as T7 RNA polymerase can be used.
  • any of the mutant TyrRSs of the present invention can be used.
  • the use of a mutant TyrRS having an enhanced aaRS activity using a tyrosine derivative as a substrate is advantageous for the production of a polypeptide incorporating a tyrosine derivative, and further has a mutant TyrRS having an increased aminoacylation reaction rate to an amber suppressor tRNA.
  • the use of is advantageous because the production efficiency can be increased.
  • the sublesser tRNA of (3) is a tRNA T mutant derived from archaebacteria or eukaryotes, and is preferably an amber suppressor tRNA (the first letter G of the tyrosine anticodon GUA is replaced with C, and the anticodon Has become CUA).
  • M. 'a / w? // Derived tyrosine sublesser tRNA can be prepared by the method described above. Eukaryotic tRNAs that can be used are described, for example, in M. Sprintl et al., Nucleic Acids Research, Vol. 17, pp. 117, 1989. Things.
  • polypeptide into which the tyrosine derivative is incorporated is not limited, and may be any expressible polypeptide or a heterologous recombinant polypeptide.
  • a nonsense codon an amber codon when the suppressor RNA is an amber suppressor
  • this nonsense codon is introduced.
  • Part Can specifically incorporate an unnatural amino acid.
  • a well-known method can be used to introduce a mutation into a polypeptide in a site-specific manner, and is not particularly limited.
  • the polypeptide is intended to have any number of residues ranging from a small peptide to a large peptide as defined above, and includes known or novel polypeptides.
  • DNA or RNA encoding the polypeptide of interest can be obtained from eukaryotic or prokaryotic cells or tissues as genomic DNA or mRNA by well-known methods (phenol Z-cloth form treatment, ethanol precipitation, cesium chloride density gradient centrifugation, etc.) Or synthesized or isolated by cDNA cloning.
  • the amino acid sequence of the polypeptide or the nucleotide sequence encoding it is known, it can be chemically synthesized using a DNA synthesizer.
  • reaction conditions such as temperature and stirring speed can be used depending on the type of polypeptide.
  • the temperature is usually about 25 to about 50 ° C, preferably about 37 ° C.
  • the shaking speed or the stirring speed can be low, for example, 100 to 200 rpm.
  • the reaction time can be appropriately selected while monitoring the production of the desired polypeptide.
  • Any cell extract preferably prokaryotic bacteria extract,
  • kits for producing a polypeptide incorporating a tyrosine derivative comprising: Further, the kit for producing a polypeptide incorporating a tyrosine derivative is described in 1 to 3. in addition,
  • Liponucleotides such as ATP, GTP, CTP, UTP
  • kits As each component of these kits, the same components as described in the cell-free polypeptide synthesis system can be used.
  • kits can easily express DNA or mRNA encoding the desired polypeptide in which an ampa mutation has been introduced at a desired position, and can thus be expressed at any desired position. It can be used for the production of a desired polypeptide incorporating a tyrosine derivative.
  • the invention also provides a method for producing a polypeptide, which is characterized by the following.
  • Escherichia spp. for example, Escherichia coli 12 strain MM294 (ATCC 31446); Escherichia coli X1776 (ATCC 31537); Escherichia coli W3110 strain (ATCC 27325); and K 5 7 7 2 (ATCC 53635); enterobacteria, Erwinia, Klebsiella, Proteus, Salmonella, enterobacteria such as Salmonella typhimurium, Serratia, and Shigella, B. subtilis, B. Includes, but is not limited to, Bacillus such as licheniformis, Pseudomonas such as Pseudomonas aeruginosa, and Streptomyces.
  • the method can be performed according to a method in which suppressor tRNA is expressed.
  • eubacteria having This eubacteria can be used in the method for producing a polypeptide described above.
  • the tyrosine derivative-incorporated polypeptide produced in Escherichia coli using the TyrRS of the present invention can be recovered from a culture medium or a host cell lysate by a conventional method. If attached to a membrane, it can be released from the membrane using a suitable detergent (eg, Triton-XI00) or by enzymatic cleavage. Cells can be disrupted by various physical-chemical means such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.
  • the protein denaturing agent when solubilizing the insoluble substance with a protein denaturing agent, the protein denaturing agent is not contained or the concentration of the protein denaturing agent is so low that the protein is not denatured. It can be diluted or dialyzed to form a protein tertiary structure.
  • Isolation and purification of polypeptides include solvent extraction, fractional precipitation with organic solvents, salt precipitation, dialysis, centrifugation, ultrafiltration, and ion exchange chromatography, based on the specific properties of the produced polypeptide. Separation operations such as gel filtration chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, crystallization, and electrophoresis can be performed alone or in combination.
  • the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
  • PCR was performed under the following conditions.
  • the temperature control of the reaction was as follows.
  • y y is 30 or more, and the length of the DNA fragment generated by PCR is divided by 500 and multiplied by 60.
  • pET-YRS Escherichia coli BL21 star
  • E3 Escherichia coli minor codon complement plasmid pRARE
  • the above E. coli transformed with PET-YRS was cultured in LB medium 2/100 g / m / ampicillin. 0.0. 6. .
  • IPTG isopropyl-1-thio-D-galactopyranoside
  • the obtained supernatant was crystallized by two-step column chromatography using an anion exchange column Q Sepharose Fast Flow (Amersham Biosciences) and an affinity column HiTrap Hepar in HP (Amersham Biosciences). Purified to a purity suitable for About 30 mg of a purified sample was obtained per 1 / culture liquid.
  • the amount of transcript of tRNA T of bacterium is small as it is because the 5 'end is a cytidine residue.
  • tRNA transcribed in T7RNA polymerase as a first hammer one Heddoripozaimu fusion (transzyme), it was decided to create a tRNA Ty r by subsequent self-cleavage.
  • Ddoripozaimu to hammer 5 'end of the NA Tyr coding region creating a DNA having a sequence 27 that transcription promoting sequence was added in the bonding of synthetic primer, to introduce it into pUC119.
  • plasmids were prepared in large quantities.
  • the obtained plasmid was used as a type II, and a T7 RNA polymerase transcription reaction was performed to synthesize transzyme RNA.
  • the self-cleavage reaction of the hammerhead lipozyme was promoted by performing 12 to 14 cycles of transzyme at 85 ° C for 30 seconds and 60 ° C for 9 minutes.
  • tRNA T is purified and deurea purified by 8 M urea-15% polyacrylamide gel electrophoresis and column chromatography using an anion exchange column Resource Q (Amersham Biosciences) to obtain a sample suitable for crystallization. Obtained.
  • the purified tRNA Tyf was dissolved in 20 mM Tris -Cl ( H7.5 ) and 20 mM magnesium chloride solution, denatured at 80 ° C, and cooled slowly to form a higher-order structure.
  • the purified TyrRS solution was purified by ultrafiltration using Vivaspin 2 (Vivascience) in 20 mM Tris-Cl (pH 7.9), 20 mM magnesium chloride, 2 mM L-tyrosine, 10 mM 2 -The solution was replaced with a mercaptoethanol solution and concentrated.
  • the obtained solution was used as a sample for crystallization. Crystallization of the complex
  • crystallization buffer [30% (v / v) 1,6-hexanediol, 50 mM sodium acetate (pH 4.0), 200 mM ammonium chloride] , 10 mM sodium chloride and I mM zinc acetate]
  • crystallization buffer As the reservoir solution, a crystallization buffer in which the 1,6-hexanediol concentration was changed to 35% (v / v) was used. After equilibrating at 30 ° C for 1 week, hexagonal bipyramidal crystals appeared and grew to 0.15X0.15X0.45 mm after about 3 weeks (Fig. 19).
  • Diffraction data was collected at beamline BL41XU at SPring_8, a high-intensity synchrotron radiation facility.
  • the crystals were immersed in a crystallization buffer containing 30% (v / v) ethylene glycol as a cryoprotectant and then instantaneously cooled to 100K. While keeping the temperature at 100 K, diffraction data were collected for native and SeMet conjugates at an oscillation angle of 1 ° and an exposure time of 2 seconds (Fig. 20).
  • the X-ray absorption fine structure (XAFS) spectrum was measured to enhance the anomalous dispersion effect of Se atoms, which is essential for phase determination, and the absorption edge wavelength (0.9793A (0.09793nm)) was measured. The diffraction data was measured. The measured XAFS spectrum is shown in Figure 21.
  • the initial phase of the SeMet-labeled complex was analyzed using the single-wavelength anomalous dispersion (SAD) method.
  • the initial coordinates of Se atoms were determined by the direct method using the SnB program based on the scaling data of the SeMet-labeled complex.
  • MLPHARE of the Collaborative Co. utational Project No.4 (CCP4) program suite
  • CCP4 Collaborative Co. utational Project No.4
  • RESOLVE program to extend the phase to 2.1 A (0.21 nm) and improve the electron density by solvent smoothing and histogram matching, a clearly interpretable electron density was obtained.
  • Figure 22 shows the difference Fourier diagram for the abnormal variance.
  • the model was constructed using program 0.
  • the model of the constructed complex was refined by using program CNS to refine the temperature factor of each atom and repeating simulated annealing. Of the total data, 10% selected at random is excluded from refinement for cross-validation.
  • the aminoacylation reaction was performed at 37 ° C.
  • the composition of the reaction solution was 100 mM HEPES-KOH (H7.5), 15 mM magnesium chloride, 0.05 mg / m spore serum albumin, 1 mM dithiothreitol, 10 mM ATP, and 12 L_ [ 14 C] -tyrosine solution.
  • the enzyme concentration in the reaction solution was set to 10 nM or 100 nM, and the initial reaction rate when the tRNA concentration was changed from 0.25 to 64 M was determined from the time change of the radioactivity of the acetic acid-precipitated trichloride fraction. The kinetic constants were determined by plotting.
  • the asymmetric unit contained one TyrRS subunit, a tRNA Tyr , and a tyrosine molecule.
  • TyrRS forms a homodimer as suggested by the gel filtration method, and the two units in the homodimer are related to each other with a twofold symmetry axis (Fig. 23).
  • Fig. 6 shows the overall structure of the triple complex
  • Fig. 8 shows the electron density near the tyrosine binding site.
  • a part of the KMSKS loop (residues 203 to 209) is disordered because ATP, one of the substrates of TyrRS, is not in a bound state.
  • the nucleotide residues of the tRNA Tyr of the complex have a stable structure except for the 3 'end.
  • the two tRNA Tyr molecules span the two subunits of the homodimer, the receptor stem of the tRNA is in one subunit, and the anticodon loop interacts with the other subunit.
  • T./TyrRS has an additional C-terminal domain after the anticodon binding domain, which recognizes a long variable arm characteristic of eubacteria (Fig. 9 (d)).
  • ⁇ The tRNA bound to / ⁇ / / TyrRS is approximately 18 compared to that of M. / 3/7 /? ( ⁇ //. (Fig. 11) tRNA receptor recognition
  • the CI: G72 base pair of the Axepyu stem is the most important factor for the orthogonality of the TyrRS.tRNA T pair.
  • the except stem is recognized by the Rossmann old domain and the CP1 domain (Fig. 12).
  • G72 base The minutes are parallel to the other base planes of the acceptor stem, whereas the bases of C1 are twisted about 20 ° and tilted further 20 ° ( Figure 13).
  • the C1 base in this twisted position is strictly recognized by the enzyme.
  • the carbonyl group at position 2 and the nitrogen atom at position 3 of the C1 base are hydrogen-bonded to the guanidino group of Argl74, and the amino group at position 4 is hydrogen-bonded to the water molecule captured by Argl32. It also has hydrophobic interaction with the side chain of Metl78 ( Figure 13). G72 is mainly recognized by the amino group on the side chain of Lysl75. Argl32, Argl74, and Lysl75 are highly conserved among germs and eukaryotic TyrRS ( Figure 2). Rigorous recognition of C1 is thought to be the basis for C1: G72 specificity in TyrRS. In fact, M.
  • a / w // TyrRS is, Ul: the A72 base was converted pairs into the yeast M Ty r mutants, only recognize an efficiency of 1/200 as compared with the wild-type tRNA TYF (Reference 1 8) .
  • A73 is also a strong identity determinant of tRNA, and if it is replaced with guanine or peracil, it will not be recognized by zo aw // TyrRS (Reference 18).
  • the amino group at position 6 and the nitrogen atom at position 1 are recognized by the carbonyl and amino groups in the main chain of Vall95, respectively (Fig. 13).
  • Argl32 recognizes G71
  • Thrl26 and Glul82 recognize the tRNA phosphate backbone (Fig. 12).
  • Gl C72 is recognized in a different manner in T. thermophilus 1 ? & (Fig. 14). Not much is known about G1, only C72 is recognized by one hydrogen bond with Glul54. M. a / w? TyrRS does not have a proton axel in the corresponding part (Fig. 13). Furthermore, it was clarified that the A73 base that had been thrown from the helix axis in the. Complex was located on the extension of the helix in ⁇ . TlwrnwpIiUus (Figs. 13 and 14).
  • TyrRS of archaebacteria and eubacteria recognize axepstein stems with completely different residues, despite similar backbone structures.
  • G1 In the case of a tRNA with C72 base pairs. In jannaschii TyrRS, the functional group of Gl is far away from the side chain of Argl 74, and there is no proton receptor that can form a hydrogen bond with the amino group at the 4-position of C72. Is not considered. In fact, biochemical analysis supports this (Reference 18). On the other hand, T./force-force// does not recognize C1: G72 base pairs.
  • anticodons are recognized by the C-terminal domain ( Figure 6).
  • G34 the first letter of the anticodon, is derived and strictly recognized ( Figure 15).
  • the base part of G34 is stacked between the rings of Phe261 and His283, and the nitrogen atom at position 1 and the amino group at position 2 are both recognized by hydrogen bonding with Asp286.
  • the aminoacylation rate of the D286A substitution product is 1/10 or less of that of the wild type.
  • the other bases of the anticodon do not interact with the protein in a very specific manner.
  • the nitrogen atom at position 3 of U35 is a carbonyl group in the main chain of Cys231, and the report of A36 is the main chain of Lys288 and Lys228. It is only interacting with the chain (Fig. 15).
  • the phosphate group of C28 is hydrogen bonded to the side chain of Lys228. This is considered to be the reason why the first letter of anticodon is distinguished much more strongly in zoam / TyrRS than in other bases (Reference 18).
  • Asp286 is highly conserved in archaeal and eukaryotic TyrRS, and corresponds to the aromatic residue corresponding to Phe261 on the / 3-31 () -3 motif (Fig. 10) and to His283. Histidine residues are also well conserved ( Figure 2). For example, in human TyrRS, T ⁇ 283, Asp308, and His305 correspond to Phe261, Asp286, and His283 of ⁇ awz7 TyrRS on the primary sequence, respectively (FIG. 2). Furthermore, the orientations of Trp286 and Asp308 side chains on the three-dimensional structure of human TyrRS are almost the same as those of Phe261 and Asp286 of M. zoa /? // TyrRS.
  • T. thermophilus ⁇ recognizes anticodons by a completely different mechanism than M. zoaz / zc force // TyrRS.
  • G34 is screened at A36, and the modified base, pseudouridine residue 35, is inverted ( Figure 16). And G34 is jannaschii x ⁇ on the primary structure The carbonyl group is recognized by Asp259, which does not correspond to Asp286 (Fig. 2, 16).
  • Pseudouridine residue 35 is primarily recognized by Asp423, but the residue belongs to the C-terminal domain unique to eubacteria TyrRS, and no corresponding residue exists in archaeal / eukaryotic TyrRS ( Figures 2 and 16). Modification of anticodon recognition based on conformation
  • Asp286 of ⁇ azoa; ?? 7 '/ TyrRS is a key residue for specifically recognizing G34, which is the first letter of the anticodon. Therefore, in order to increase the efficiency of aminoacylation of the tRNA T G34C mutant, ambassador sub-resor tRM, a mutation was introduced at residue 286. In the case of cytosine at the 34th base of tRNA in wild-type TyrRS, even if the base is inverted as in G34, the distance between Asp286 and the base is too large to obtain satisfactory interaction. it is conceivable that.
  • mutants in which Asp286 was substituted with Glu, Phe, Ile, Leu, Gin, Arg, and Tyr, which are larger side chains, were prepared, and the activity of aminoacyl sulfide on amba-sublesser-tRNA was measured.
  • the initial reaction rate was determined at a relatively high concentration of amber suppressor tRNA, a significant increase in activity was observed for the Gln, Arg, and Tyr substitutions.
  • the D286R showed an initial velocity eight times that of the wild type (Fig. 17).
  • TyrRS wild type TyrRS (D 2 8 6 R)
  • Table 2 shows the Michaelis constant and the kinetic constant for TyrRS of the wild type and the mutant.
  • Aminoacylation activity against wild-type TyrRS in amber sable suspended one tRNA was about 1/300 as compared with the wild-type tRNA Ty r.
  • the D286R mutant was found to aminoacylate amber suppressor tRNA to the same extent as the wild-type tRNA Tyr .
  • the D286R mutant was found to recognize tRNA 65 times better than the wild-type enzyme.
  • the / i m for Anba one Saburetsusa tRNA is a mutant has been mainly reduced, ⁇ did not change much.
  • the D286R, D286Q, and 3286Y mutants obtained in the present invention are expected to improve suppression efficiency in a w> o or / or in vitro system. By combining these mutations with mutations in the amino acid binding site for specifically recognizing non-natural amino acids, it is thought that large quantities of aloprotein can be produced.
  • thermophilus and M./'/TyrRS the CCA terminus of tRNA is disordered in the three-dimensional structure, so it has not been possible to verify which is the true target.
  • the KMSKS loop that binds ATP and the CCA terminus were both out of order, so we believe that there is some relationship between the two.
  • Tyrosine is housed in a deep tyrosine binding pocket in the enzyme ( Figure 4). Near the entrance of the tyrosine binding pocket, the amino and carbonyl groups of tyrosine form a hydrogen bonding network at Glnl73, Tyr151, and G1n155 ( Figure 4). The phenyl ring of tyrosine is recognized by the side chains of Leu 65, His 70, and G1n155, and further by the main chains of I1e33, G1y34, Phe35. Recognized (behind tyrosine, not shown).
  • hydroxyl group on the side chain of tyrosine is recognized by hydrogen bonding at Tyr32 and Asp158 inside the pocket ( Figure 4).
  • Water molecules are hydrogen-bonded by His 1 7 7 and Ty r 3 2 And is also proximal to the tyrosine side chain ( Figure 4).
  • Tyrosine-free human mini-TyrRS also has this type of amino acid binding pocket.
  • archaeal and eukaryotic TyrRS have conserved tyrosine binding sites.
  • Residues in the tyrosine binding pocket are also well conserved in bacteria.
  • the arrangement of the residues that form the deep pocket is similar to the B. stearothernwphilus and S. aureus TyrRS structures. jannaschim yr32, Aspl58, Glnl55, G1n173, and Tyrl515 in B. stearothennophi lus! corresponds to yr34, Aspl76, Gln173, Gln95, and Tyr169.
  • T. ihermophuIus y ⁇ RS has an exceptional Lys in place of Tyr to recognize the tyrosine hydroxyl group, but other residues in hydrogen bonding to tyrosine are conserved .
  • the details of the tyrosine binding site of TyrRS differ between archaeal eukaryotes and bacteria.
  • ⁇ is 70 and H s 177 of M. iannas chiii TyrRS are absent in A force // TyrRS.
  • the configuration of residues that do not directly interact with tyrosine is ⁇ : in M. jannaschii and B. stearothermophi lus.
  • the side chain of As n 123 is about 4 A (0.4 nm) from the tyrosine hydroxyl group of B./ea/o/force e3 ⁇ 4o 7i «TyrRS, but the corresponding residue of TyrRS of M.zo 3? /?
  • the group G 1 u 107 is 13 A (1.3 nm) away from tyrosine.
  • the Y32Q and D158A mutations are required to form a p-methyl-L-tyrosine binding pocket.
  • the E107T and L162P mutations appear to be either naturally or indirectly affected mutations because they are not close to binding tyrosine.
  • Stearo1 ⁇ 2er 3 ⁇ 4? Computer with Ja / jas / TyrRS structure based on 7i / sTyrRS structure — Gluul 07 and Leul 62 are located away from the tyrosine bond ⁇ , and indirectly L— It has also been shown to contribute to the elimination of tyrosine.
  • FIG. 4 it is possible to obtain new information on the details of the amino acid binding site for more effectively creating the unnatural amino acid specificity.
  • a D286R mutant that efficiently recognizes amber suppressor-tRNA (C34G mutant of tRNA T5T ) was obtained. Since this mutant has a low ⁇ to suppressor-tRNA, it may be possible to co-crystallize with the tRNA.
  • variants that specifically recognize various unnatural amino acids have been obtained (References 4, 5, 11, 13 and 24). In the structure of the complex determined this time, it was revealed that the recognition mechanism of the tyrosine side chain at the tyrosine binding site was similar to that of the previously determined structure of stearothermophilus TyrRS.
  • the determination of the structure of the M.; masc complex allows modification based on the structural information of the substrate binding site and the tRNA binding site.
  • the structure of stearothermophilus TyrRS was used to infer the structure of the substrate binding site, but the arrangement of the fine residues was different.
  • Glul07 of M. J'a / 3 / zasdz TyrRS has the primary sequence B. It corresponds to Asnl23 of TyrRS, but B.
  • V 6A 00 " ⁇ 86 • on HI ⁇ 8 • ⁇ f 6 V 3W 03 ⁇ V
  • V 6S 200 "I m • ⁇ 668 • 6Z no 01 V NSV 98 Thigh V
  • V U "92 00 • i 210 • 86 999 • OS 688 • is u V V3 ozt WOXV
  • V 89 • 88 00 ' ⁇ l ⁇ • 001 • ⁇ 8 Z86 • is ⁇ V ao 861 Thigh V
  • V 29 ⁇ 0 00 ' ⁇ • 36 9A0 • is V mo 3 WOXV
  • V 21 • ⁇ 00 952 • 801 ⁇ 6 ⁇ 6 ⁇ 86S 'l ⁇ V SIH 90 6SS ⁇ V
  • V L • ii 00 ' ⁇ "801 0 6' 9 ⁇ • is 8 V ⁇ 3 26S
  • V 88 '92 00 ' ⁇ 0Z9 • zu 96Z O 991 ⁇ is 9 ⁇ V UL iao 8ZS Marauder V
  • V 06 ⁇ 00 • ⁇ m • 96 980 '9 S9 V Thigh 0 ZCS ⁇ V
  • V 08 • is 00 • i 916 '86 9 996 88 V m 33 plate V
  • V 8 • OS 00 ⁇ ⁇ 888 "001 100 S 8St '11 38 V an V3 069 Thigh V
  • V l ⁇ n 00 • t 898 • 801 'IS • is 16 V sn 09 i thigh V

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Abstract

L'invention concerne un mutant TyrRS ayant une séquence aminoacide dérivée de la séquence aminoacide représentée par SEQ ID NO :1, par substitution d'un ou de plusieurs restes aminoacides dans « Tyr32, His70 et Asp58 » ou d'un ou de plusieurs restes aminoacides dans « Tyr32, Asp158 et His177 » par un autre, ou plusieurs autres restes aminoacides, et ayant une activité aminoacyl t-ARN synthase accrue, en utilisant la 3-iodotyrosine comme substrat, comparativement à l'activité aminoacyl t-ARN synthase en utilisant la tyrosine comme substrat ; ou un mutant TyrRS ayant une séquence aminoacide avec substitution de Asp286 par un autre reste aminoacide et ayant une vitesse d'aminoacylation élevée, à un suppresseur t-ARN, comparativement au TyrRS comprenant la séquence aminoacide représentée par SEQ ID NO :1.
PCT/JP2004/001441 2003-02-10 2004-02-10 Mutants de tyrosyl t-arn synthase, et procede de construction de ceux-ci WO2004070024A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008001947A1 (fr) 2006-06-28 2008-01-03 Riken SepRS MUTANTE, ET PROCÉDÉ D'INTRODUCTION SITO-SPÉCIFIQUE D'UNE PHOSPHOSÉRINE DANS UNE PROTÉINE EN UTILISANT CETTE SepRS
EP1961817A1 (fr) * 2005-11-24 2008-08-27 Riken Procede pour la production de proteine renfermant un acide amine de type non naturel
WO2009038195A1 (fr) 2007-09-20 2009-03-26 Riken Pyrrolysyl-arnt synthétase mutante, et procédé l'employant pour produire une protéine dans laquelle sont intégrés des acides aminés non naturels
WO2023282315A1 (fr) 2021-07-07 2023-01-12 味の素株式会社 Procédé de production sécrétoire d'une protéine contenant un acide aminé non naturel
CN115717130A (zh) * 2022-09-02 2023-02-28 凯莱英医药集团（天津）股份有限公司 氨酰-tRNA合酶突变体及烯基酪氨酰-tRNA的制备方法
US11827684B2 (en) 2020-04-22 2023-11-28 Merck Sharp & Dohme Llc Human interleukin-2 conjugates biased for the interleukin-2 receptor beta GAMMAc dimer and conjugated to a nonpeptidic, water-soluble polymer

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EP2246428A4 (fr) * 2008-02-07 2011-04-13 Univ Tokyo Arnt modifié contenant un nucléotide non naturel et son utilisation
KR102655597B1 (ko) * 2021-06-14 2024-04-08 전남대학교 산학협력단 티로실-tRNA 합성효소 돌연변이체 및 이를 이용한 4-아지도-L-페닐알라닌의 결합능이 향상된 단백질 생산방법

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1961817A1 (fr) * 2005-11-24 2008-08-27 Riken Procede pour la production de proteine renfermant un acide amine de type non naturel
EP1961817A4 (fr) * 2005-11-24 2009-07-15 Riken Procede pour la production de proteine renfermant un acide amine de type non naturel
US8183013B2 (en) 2005-11-24 2012-05-22 Riken Method for production of protein having non-natural type amino acid integrated therein
US8642291B2 (en) 2005-11-24 2014-02-04 Riken Method for producing proteins comprising non-natural amino acids incorporated therein
WO2008001947A1 (fr) 2006-06-28 2008-01-03 Riken SepRS MUTANTE, ET PROCÉDÉ D'INTRODUCTION SITO-SPÉCIFIQUE D'UNE PHOSPHOSÉRINE DANS UNE PROTÉINE EN UTILISANT CETTE SepRS
JP5305440B2 (ja) * 2006-06-28 2013-10-02 独立行政法人理化学研究所 変異体ＳｅｐＲＳ及びこれを用いるタンパク質への部位特異的ホスホセリン導入法
WO2009038195A1 (fr) 2007-09-20 2009-03-26 Riken Pyrrolysyl-arnt synthétase mutante, et procédé l'employant pour produire une protéine dans laquelle sont intégrés des acides aminés non naturels
US8735093B2 (en) 2007-09-20 2014-05-27 Riken Mutant pyrrolysyl-tRNA synthetase, and method for production of protein having non-natural amino acid integrated therein by using the same
US9133449B2 (en) 2007-09-20 2015-09-15 Riken Mutant pyrrolysyl-tRNA synthetase, and method for production of protein having non-natural amino acid integrated therein by using the same
US11827684B2 (en) 2020-04-22 2023-11-28 Merck Sharp & Dohme Llc Human interleukin-2 conjugates biased for the interleukin-2 receptor beta GAMMAc dimer and conjugated to a nonpeptidic, water-soluble polymer
WO2023282315A1 (fr) 2021-07-07 2023-01-12 味の素株式会社 Procédé de production sécrétoire d'une protéine contenant un acide aminé non naturel
CN115717130A (zh) * 2022-09-02 2023-02-28 凯莱英医药集团（天津）股份有限公司 氨酰-tRNA合酶突变体及烯基酪氨酰-tRNA的制备方法

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